Molecular Mechanism Of Transcription Factor ZmNAC20 Regulating Maize Response To Drought Stress

Maize,one of the world’s top three food crops,is also one of the most widely grown crops in the world.However,its production is seriously threatened by drought stress,and continuous selection and breeding of new varieties that are more resistant to drought is one of the effective ways to solve this problem.The rapid development of new biotechnologies such as transgenics has greatly improved the breeding efficiency,and it is important to explore the drought resistance genes and study their mechanisms of action,and to use transgenic technology for crop improvement to breed new drought resistant varieties of maize.In this study,we identified a NAC gene,ZmNAC20,that is associated with adversity stress and its expression was predicted to be related to abscisic acid(ABA)and drought stress by bioinformatics analysis of the promoter element of ZmNAC20.Real-time fluorescence quantitative PCR(q RT-PCR)revealed that ZmNAC20 was induced by ABA and drought stress to up-regulate its expression.To test whether ZmNAC20 is involved in drought stress response,we constructed a ZmNAC20 overexpression vector and obtained overexpressed maize plants(ZmNAC20-OE)by transferring the vector into maize self-incompatibility line B104 through genetic transformation technique.By drought stress treatment,ZmNAC20 overexpression was found to enhance drought resistance in maize.The specific findings were as follows:(1)ZmNAC20 expression was induced up-regulated by ABA and drought stress.After dehydration stress treatment of maize seedlings that grew for 12 days,q RT-PCR analysis showed that the expression of ZmNAC20 in maize leaves was significantly upregulated,indicating that drought stress promoted the expression of ZmNAC20.Maize leaves treated with different concentrations of ABA for 1 h showed that ABA treatment increased the expression of ZmNAC20.Treatment with 1 μM ABA for different times also promotes upregulation of ZmNAC20 expression.These results suggest that drought stress and ABA jointly regulate the expression of ZmNAC20.In addition,treatment of maize plants with PEG and salt stress could also induce ZmNAC20 upregulation expression.ZmNAC20 was found to be localized in the nucleus using laser confocal microscopy.At the same time,the promoter element analysis of ZmNAC20 gene showed that the upstream promoter region contained cisacting elements related to drought and ABA response.(2)ZmNAC20 overexpression enhances drought resistance in maize.In order to explore the function of ZmNAC20 gene in maize response to drought stress,ZmNAC20 overexpression vector was genetically transformed to maize inbred line B104,and ZmNAC20 overexpressed ZmNAC20-OE was obtained.After 14 days of drought stress,ZmNAC20-OE maize leaves were slightly yellow and curled,while the control B104 maize leaves began to wilt or even die.After 3 days of rehydration,the leaves of ZmNAC20-OE maize gradually turned green,and the survival rate reached about 80%,which was much higher than that of B104,indicating that ZmNAC20-OE maize plants had obvious drought resistance.During 12 days of drought stress,the activity of superoxide dismutase(SOD)and peroxidase(POD)and the ability to remove reactive oxygen species in ZmNAC20-OE maize plants were significantly higher than those of B104,indicating that ZmNAC20 overexpression under drought stress could affect the antioxidant enzyme system and the accumulation of reactive oxygen species in maize plants,thereby affecting the drought tolerance of plants.ZmNAC20-OE maize had higher relative leaf water content under drought stress,indicating that ZmNAC20-OE maize plants could maintain normal cell growth for a longer time under drought stress,and had a positive effect on improving drought resistance.In addition,ZmNAC20-OE maize leaves had stronger photosynthetic efficiency and biomass accumulation under drought stress.These results show that ZmNAC20 can reduce the harm caused by drought stress to maize and improve the ability of maize to resist drought stress.(3)ZmNAC20 overexpression promotes ABA-mediated stomatal closure.In order to explore the relationship between maize leaf stomata and ZmNAC20 gene drought resistance,this study observed the changes of stomatal movement of B104 and ZmNAC20-OE maize under drought stress,and found that after 200 min of dehydration stress treatment,the maize leaves of B104 were severely curled,while the leaves of ZmNAC20-OE maize showed a slight curl phenotype,and the water loss rate of ZmNAC20-OE maize leaves was lower than that of B104.These results showed that ZmNAC20-OE maize leaves had stronger resistance to dehydration,and ZmNAC20 overexpression could promote maize leaves to retain more water and enhance drought resistance.The results of stomatal observation showed that under dehydration stress,ZmNAC20-OE maize leaf stomata closed more rapidly than B104,with less water dispersion loss.The stomatal closure speed of ZmNAC20-OE maize under ABA treatment was faster than that of B104,and the sensitivity of ZmNAC20-OE plant stomatal closure to ABA was higher than that of B104.These results show that ZmNAC20 promotes maize stomatal closure and reduces water loss,thereby enhancing maize drought resistance.In addition,under drought stress,ZmNAC20 can also induce the expression of ABA biosynthetic genes such as ZmNCED3 and stomatal development related genes such as Zm SLAC1.(4)ZmNAC20 activates the expression of multiple stress-responsive genes.RNA-Seq analysis was used to show that the up-regulated genes of ZmNAC20 overexpressed plants after dehydration stress treatment were mainly enriched in abiotic stimulation,photosynthesis,hormones,cold,water shortage,salt stress and osmotic stress,and ZmNAC20 significantly promoted the upregulation and expression of Zm DHN2,Zm GA2OX6 and Zm SWEET17 A and other stress response genes under drought stress and ABA treatment.These results show that ZmNAC20 regulates the expression of various genes in drought stress response,and ZmNAC20 improves drought resistance through a variety of gene regulatory networks.(5)ZmNAC20 is involved in regulating multiple signal transduction pathways.Using DAP-seq technology,it was found that ZmNAC20 directly binds to 1304 target genes.The sequences directly bound to ZmNAC20 were analyzed and it was found that the bound fragments were mainly concentrated near the transcription initiation site.Since transcription factors regulate transcription by identifying specific DNA sequences,analysis of significant element sequences in ZmNAC20 binding fragments revealed 3 very significant core binding elements.Through KEGG analysis,it was found that ZmNAC20 was mainly involved in regulating RNA transport,hormone signal transduction,MAPK kinase signal transduction and other pathways.Under drought stress,65 target genes directly regulated by ZmNAC20 were found through DAP-seq and RNA-seq association analysis,of which 43 were upregulated and 22 were downregulated.Based on the above results,this study identified the transcription factor ZmNAC20 as drought resistant in maize.It improved the drought tolerance of maize by promoting stomatal closure and the expression of activated stress genes,and revealed the molecular mechanism of plant adaptation to water deficit stress.At the same time,it provides a valuable gene and new clue for the response mechanism of maize to drought stress,which is of great significance for the genetic improvement and molecular breeding of maize drought resistance.